The study characterized Exserohilum turcicum, the causal agent of Turcicum Leaf Blight (TLB) in sorghum, isolated from different regions of Tharaka Nithi County, Kenya. Six isolates typical of E. turcicum were obtained from symptomatic leaf samples. The isolates varied in colony morphology, pigmentation, growth patterns, and conidia size. Conidia length ranged from 71.32 to 200.83 μm and was significantly different among isolates. Microscopic analysis found septate, branched mycelium and simple, cylindrical conidiophores bearing solitary conidia. Variations were observed, suggesting morphologically different TLB pathogens in the county.
Morphological characterization of sorghum e. turcicum isolate from different areas of tharaka nithi county, kenya
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Journal of Scientific and Engineering Research, 2018, 5(12):218-226
Research Article
ISSN: 2394-2630
CODEN(USA): JSERBR
Morphological Characterization of Sorghum E. turcicum Isolate from Different Areas
of Tharaka Nithi County, Kenya
Fredrick O. Ogolla*1
, Benson O. Onyango3
, Moses M. Muraya2
, Sioma Mulambula2
1
Department of Biological Sciences, Chuka University, P.O. Box 109-60400, Chuka, Kenya
2
Department of Plant Sciences, Chuka University, P.O. Box 109-60400, Chuka, Kenya
3
Department of Biological Sciences, Jaramogi Oginga Odinga University of Science and Technology, P.O. Box
210 – 40601, Bondo – Kenya
Abstract Low sorghum production in Kenya is attributed to devastation by TLB. Reaction similar to those of
TLB have been observed in different farms in Tharaka Nithi County with varied lesion sizes. However, little
scientific attention has been expended to characterize the pathogen and provide knowledge on TLB pathogen
occurrence in the area. The study was conducted to characterize E. turcicum pathogen from different regions of
Tharaka Nithi County in Kenya using cultural and morphological characteristics. Pathogen isolation from
symptomatic leaves collected between April and July 2018 was done at Chuka University microbiology
laboratory. Six out of nine samples collected from different regions of Tharaka Nithi county were typical E.
turcicum. Data on conidia area was subjected to one-way analysis of variance using SAS software version 9.3
and LSD used to separate significantly different means at 5% probability level. There was statistically
significant difference on conidia length among E. turcicum isolates (P<0.05). The conidia length and width for
isolates (K005, T002 and G001) were within those already documented range. However, isolates G002 and
M004 recorded longer length of up to 200.83 and 184.63um respectively. Mycelium appeared septate, branched
and brownish while the Conidiophores was simple, cylindrical and septate. Variations were observed on isolates
pigmentation, growth pattern and conidia sizes. The results show that morphologically different TLB pathogen
are responsible for causing the disease in Tharaka Nithi County. Molecular characterization is recommended to
provide information on genetical variability of TLB pathogen in Tharaka Nithi county.
Keywords E. turcicum, Morphological, Characterization, Tharaka-Nithi, Kenya
Introduction
Turcicum leaf blight of sorghum is caused by the heterothallic ascomycete Exserohilum turcicum the pathogen
formerly known as Helminthosporium turcicum [1-2]. It also causes northern corn leaf blight (NCLB) on maize
and related wild grasses [3] Turcicum Leaf Blight is among the most destructive foliar diseases of maize and
sorghum [4]. Exherohilum turcicum has been reported to produce non-specific toxic compounds in culture [5]
[6] as well as cultivar-specific toxin [7]. The efficacy and production of these toxins greatly depend on
temperature and pH [6,8]. Enzymatic and toxic activities of the fungus accounts for the pathogenicity [6]. The
susceptible plant varieties such as sorghum experiences physiological challenges which include their capacity to
prevent infection if inoculated with the pathogen [4]. Infection of plant’s leaves by TLB leads to reduced green
leafy area, increased leaf transpiration, limited translocation and uptake of essential plant nutrients to affected
leaves and plant cells [4]. Yield reductions due to TLB can be significant depending on disease severity, timing,
and plant susceptibility [9]. Although symtoms similar to E. turcicum have been observed in a prelimianry
investigation in sorghum plantas in Tharaka Nithi County in Kenya, little scientific attaention has been given to
isolate and characterize the pathogen.
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Exserohilum turcicum belongs to the division Eumycota, subdivision Deuteromycotina, Class Deuteromycetes,
order Moniliales and family Demetiaceae. It is a polycyclic, heterothallic and facultative parasite with three
distinct mating types: MAT 1, MAT 2 and MAT 1, 2 [10]. The fungus overwinters as mycelia, conidia and
chlamydospores in infected leaves, husks and other plant parts left on the soil surface providing innoculum for
primary infection [11]. The disease is characterized by long spindle shaped necrotic lesions on leaves measuring
up to 12.5 x 2.2 cm [12]. Pseudothecia are black, globose to ellipsoidal, 40–56 × 12–15 µm with clearly visible
setae around the ostiole. Asci are cylindrical, clavate, short pedicellate and bitunicate and 1–8 ascospores [10].
Conidia of the fungus are olive grey and spindle shaped, curved, elongated and measuring 5 x 20 μm with one
to nine septa [13]. Conidiophores are simple, cylindrical and olivaceous brown [14]. Germination of E.
turcicum conidia is bipolar and occurs 3-6 hours after inoculation. Germ tubes are 20-150 μ long and in general,
grow at an angle rather than parallel to the veins of the leaf [15]. It has a single conidium formed terminally on
the conidiophore, which resumes growth to produce new conidium at the new tip [12]. Conidia has a hilum that
protrudes distinctly from the conidia to bluntly rounded basal cells useful for identification [1]. Circular conidial
scars are evident on the conidiophore after the abscission of the conidia [1, 14].
E. turcicum isolates show variation in; colony character, colony diameter, mycelial dry weight, spore
germination, mean length and conidia width [10]. However, morphological characterisation of E. turcicum
affecting sorghum in Tharaka Nithi County have not been done to deterimine existance of variation.
Investigation on the variability of fungal pathogens is necessary to delineate phylogenetic relationships amongst
the local species, and to provide useful taxonomic information for disease management.
Materials and Methods
Tharaka Nithi County borders the County of Embu to the South and South West, Meru to the North and North
East, Kirinyiga and Nyeri to the West and Kitui to the East and South East. The county lies between latitude 000
07’ and 000 26’ South and between longitudes 370
19’ and 370
46’ East. The highest altitude of the county is
5,200m while the lowest is 600 m Eastwards in Tharaka. The average annual rainfall of 717 mm. The highlands
(upper zone) comprise of Maara and Chuka which receive adequate rainfall for agriculture. The semi-arid
(lower zone) covers Tharaka receiving less rainfall. The high-altitude areas have reliable rainfall. The lower
regions receive low, unreliable and poorly distributed rainfall. Temperatures in the highland areas range
between 14o
C to 30o
C while those of the lowland area range between 22o
C to 36o
C. Tharaka constituency
experiences temperatures of up to 40o
C at certain periods. The county has a bi-modal rainfall pattern with the
long rains falling during the months of April to June and the short rains in October to December [16].
Figure 1: Map of Tharaka Nithi County [17]
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Experimental Design
Cleaned glassware was sterilized at the temperature of 160o
C in hot air oven (Model Memmert UNB400) for 2
hours. Potato Dextrose Agar (PDA) medium was prepared using the following ingredients for culturing the
fungi in the laboratory; Pealed and washed Potato: 200 g, Dextrose: 20 g, Agar agar: 20 g and 1000 ml distilled
water. Peeled potato pieces were boiled in 500 ml distilled water in a 1000 ml beaker to make the pieces soft.
The extract was filtered through a double layered muslin cloth. To another 500 ml of distilled water in another
1000ml beaker, 20g of agar agar was added and melted till it got dissolved. Both the solutions were mixed in
another 1000 ml beaker into which 20 g of dextrose was added. The final volume of the medium was made up to
1000 ml by addition of sterile distilled water. The pH of the medium was adjusted to 6.8 with 1 N NaOH or 1 N
HC1 as the case may be with the pH meter. The medium was transferred into the media preparation bottles and
sterilized at 121o
C and pressure of 15 psi for 15 minutes in an autoclave (Model X280A). The media was then
transferred to the water bath for media tempering at 50o
C for 30 minutes. Antibiotic (25mg/l made up of
Asdoxin and Ampicillin of equal ratio) was incorporated in the media to inhibit bacteria contaminants. Samples
collected from different villages were grouped into two categories (Regions); Upper zones (Maara and Chuka
area including– Kanwa, Mikuu, Miranji, Gatuntu and Gituntu villages) with adequate rain; Lower zone (Tharaka
area including– Kanyiritha, Tunyai, Kithinge, Kamwati, Nkairini villages) representing semi-arid area. From
these groupings nine samples were randomly selected, four from upper zone and five from lower zone
respectively for pathogen isolation and characterization. These samples were K004 (Kamwati), MJ001 (Miranji)
M004 (Mikuu), G002 (Kanwa), G003 (Gatuntu) T002 (Tunyai), G001 (Kithinge), NK001 (Nkairini), KH001
(Kanyiritha).
Section of the leaf of about 5 mm2
(infected and healthy) parts of the leaves was cut and surface sterilized with
0.5% sodium hypochloride chloride solution for 1 minutes then washed in a series of sterile distilled water to
remove the disinfectant. The pieces were transferred to sterilized blotting paper in a Petri dish to remove the
excess water. Finally, one piece was aseptically plated on potato dextrose agar (PDA) for growth. Inoculated
plates were incubated in the incubator (Memmert TYP INB200) at room temperature and monitored for fourteen
days. After the fungus has grown, cultural characteristics such as growth pattern, colour of conidia and
pigmentation were observed. Other than cultural characteristics, conidia properties such as length, width, shape,
type of germination, type of mycelium and size, presence of hilum as well as number of segments for each
isolate were accessed using adhesive tape method under LCD Inversed Biological microscope (Model
A33.1005) after staining with methylene cotton blue.
Results
The isolates varied in colony morphology traits characteristics such as fungal colour, fungal pigmentation,
margin, mode of growth (profuse or sparse). Isolate G001 had regular margin, was grey in colour with brownish
pigmentation and had profuse growth. Isolate K005 was light and grey with regular margin and had black
pigmentation. Isolate M004 was light grey with circular margin and had golden brown pigmentation and showed
profuse growth. Isolate G002 was dark grey in colour with irregular margin with greenish pigmentation and
showed profuse growth. Isolate T002 was greyish in colour, had brownish to blackish pigmentation with
irregular margin while the growth was sparse (Table 1; Plate 1). Three isolates MJ001, NK001 and KH001 did
not show cultural and morphological characteristics of E. turcicum. The morphological characteristics of the E.
turcicum studied on potato dextrose agar medium in the laboratory are tabulated below
Table 1: Cultural characteristic observation of E. turcicum Isolates from Different Location in Tharaka Nithi
County
Fungal isolate Culture Growth characteristics Place of Collection
G001 Fungal colour: Greyish
Margin: Regular margin,
Pigmentation: Brownish
Mode of Growth: Profuse,
Kithinge
K005 Fungal colour: Grey (light and dark),
Margin: Circular
Pigmentation: Black
Mode of Growth: Profuse,
Kamwati
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G003 Fungal colour: Greyish with whitish shades
Margin: Irregular
Mode of Growth: Profuse
Pigmentation: Greenish
Gatuntu
M004 Fungal colour: Light Grey
Margin: Circular
Mode of Growth: Profuse,
Pigmentation: Golden brown
Mikuu
G002 Fungal colour: Dark Grey
Margin: Irregular
Mode of Growth: Profuse,
Pigmentation: Greenish
Kanwa
T002 Fungal colour: Greyish
Margin: Irregular
Mode of Growth: Sparse
Pigmentation: Brownish- black
Tunyai
MJ001 Fungal colour: Greyish
Margin: Irregular
Mode of Growth: Sparse
Pigmentation: Brownish- black
Miranji
NK001 Fungal colour: White
Margin: Circular
Mode of Growth: profuse
Pigmentation: colourless
Nkairini
KH001 Fungal colour: Brownish
Margin: Regular with concentric rings
Mode of Growth: Profuse
Pigmentation: Brownish- black
Kanyiritha
Plate 1: Cultural Characteristics of E. turcicum Isolates from Different Location in Tharaka Nithi County
Lengths, Width and Area of Conidia of E. turcicum Isolates
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The length of E. turcicum isolates were statistically significance (p<0.05; Table S7a). The length of the conidia
observed ranged from 71.32 um in isolate K005 to 200.83 um in isolate G002 (Table 2). The width of E.
turcicum isolates were not statistically significance (p>0.05). The smallest width of the conidia observed was
15.43um in isolate K005 while the largest width was 30.42 in isolate G002 (Table 2). The results morphological
characteristics revealed that there was statistically significant difference among conidia sizes of E. turcicum
isolates from different area of Tharaka Nithi County using morphological criteria with (P < 0.05). The largest
isolate was G002 with Means of conidia area of 6130 um while the smallest isolate was G001 with conidia area
of 2055.78 um (Table 2).
Table 2: Variation in Conidia Length, Width and Area (in µm) of E. turcicum Isolates from Different location
in Tharaka Nithi County
Isolate
ID
Conidia Length
(µm)
Conidia Width
(µm)
Area of Conidia
(µm)
G002 200.83 a 30.4167 a 6130.19 a
M004 184.63 a 27.0233 ab 4975.16 ab
K005 71.32 b 15.4300 b 3505.75 ab
T002 100.13 b 22.1500 ab 2196.01 b
G003 95.56 b 30.1100 a 2877.11 b
G001 100.01 b 27.8033 ab 2055.78 b
Mean 129.1463 24.91125 3023.6
LSD 64.223 13.911 3716.611
CV (%) 23.67241 26.58261 38.727
a
Means followed by the same letters are not significantly different at 5% probability level.
General Microscopic Observation of E. turcicum Isolates
Generally, microscopic study revealed mycelium which appeared septate, branched and greyish while the
conidiophores was simple, cylindrical and septate (plate1-VI). Conidiophores suspended conidia which
appeared singly at the tips. The conidia observed in this study were straight to slightly curved and spindle
shaped (plate1- VII), the septation in the conidia ranged from 3-7 septate and conidia had protruding hilum at
one end (Table 3; Plate I-II). Bipolarly conidial germination was observed in isolate K005 (Plate 1- IV) while
the rest of the conidia from other isolates showed unipolar germination (plate1- III).
Table 3: Microcopy Characteristics of E. turcicum Isolate from Different Location in Tharaka Nithi County
Isolate Conidia length/Width Other Microscopic Features
G001 Length; 100.04 um
Width: 27.80 um
Conidia: Straight to slightly curved, protruding hilum, 3-7septa
Mycelium: Branched, Septate, grey in colour
Conidiophore: Septate, greyish
Conidia Germination: Unipolar
G002 Length: 200.50 um
Width: 30.28 um
Conidia: Straight or slightly curved, protruding hilum, 7 septa
Mycelium: Branched, Septate, brownish to grey
Conidiophore: Septate, Dark grey
Conidia Germination: Unipolar
K005 Length: 71.32 um
Width: 15.43 um
Conidia: Straight or slightly curved, protruding hilum, 3-5septa
Mycelium: Branched, Septate, brownish to grey
Conidiophore: Septate, greyish
Conidia Germination: Bipolarly
T002 Length: 101.13 um
Width: 22.15 um
Conidia: Straight or slightly curved, protruding hilum, 3-7septa
Mycelium: Branched, Septate, brownish to grey
Conidiophore: Septate, greyish
Conidia Germination; Unipolar
M004
Length: 184.63 um
Width: 27.47 um
Conidia: Straight or slightly curved, protruding hilum7-8septa
Mycelium: Branched, Septate, brownish to grey
Conidiophore: Septate, Dark grey
Conidia Germination: Unipolar
G003
Length: 95.56 um
Width: 30.11 um
Conidia: Straight or slightly curved, protruding hilum, 7 septa
Mycelium: Branched, Septate, brownish
Conidiophore: Septate, Dark grey
Conidia Germination: not observed
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I. Conidia from isolate G002 II. Conidia from isolate G001
III. Monopolar Conidia germination in isolate T002 IV. Bipolar Conidia germination isolate K005
V. The biggest conidia observed VI. Conidiophore and mycelia isolate G002
Plate 2: E. turcicum Characteristic Conidium Exhibiting Varied Characteristics (I-IV)
vii. Conidia G002
7segments and
curved conidia
i. Ellipsoid Conidia- 6 Septa ii. Ellipsoid Conidia- 3Septa
Septum
Monopolar germination Bipolar germination
4 septa
v. Isolate M004 - biggest
Conidia
Conidiophore
Mycelia
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Discussion
Results on the morphology of E. turcicum showed significant distinctions between the six isolates cultured. The
difference was observed in colony margin, pigmentation and growth pattern (Table 1). The results are in line
with those of Rajashwal, [18] and Abdulaziz, et al., [19] that there exists variation in E. turcicum colony colour,
pigmentation and growth pattern. A study by Rajula et al., [20] on sorghum E. turcicum in Western region of
Kenya also reported similar cultural and morphological variation. The conidial shapes observed in this study
were curved, spindle or elongated with characteristic protruding hilum on one end which agreed with findings
by Sivanesan [21]; Abebe and Singburaudom, [22]; Harlapur et al. [23]; Rajeshwar et al., [4] and Rani, [14].
Bipolar and monopolar conidial germination was observed in this study on isolates K005 and T002 respectively
(Plate I-III, IV and VI). Similar observation on conidia germination had earlier been made by Rajeshwar et al.,
[4]. Isolate G002 had conidial length of 200.50 um and width of 30.50um while isolate M004 had length of
184.64um and width of 27.47um (Table 3). These conidial lengths were longer than those described on E.
turcicum by Ellis [24], Shurtleff, [25], Rani, [14], and Wani [12]. Variation on morphology of other plant fungal
pathogens have also been reported. Bunker et al., [26] observed variability in five isolates of Bipolaris maydis
from three different sites. The variations were on conidia mean, length and pathogen morphological and cultural
variations. Variation in pathogen morphology are brought about by changes in environment conditions which
may lead to mutations [22] Thus, frequent monitoring is necessary for timely detection and management of
fungal pathogen of economically important crops. Frequent monitoring will help to contain emergence of new
variants (mutants) that are likely to evade the existing control methods.
Conclusion
The results obtained from the current study proved that the E. turcicum pathogen is the causative agent of
turcicum leaf blight of sorghum in Tharaka Nithi County. The E. turcicum isolates studied were culturally and
morphologically different. The variations were observed in cultural charateristics such as colour, pigmentation,
growth and pattern. Morphologically, conidia lengths were significantly different while their width were not
significantly different. The variations observed were attributed to the prevailing varied weather conditions in
Tharaka Nithi County. The current study recommendation is that molecular characterization should be done for
race determination of E. turcicum isolates from Tharaka Nithi County since they registered variation both on
cultural and morphological aspects.
Acknowledgement
The research was partially funded by Chuka University internal research grant 2017/2018 cycle. I acknowledge
the assistance of Mr. Sam Chabari who played key role in sample collection, Ms. Truphena Koech for her
significant contribution in laboratory work. Lastly, I acknowledge all the staff at the department of Biological
Sciences, Chuka University for their consistent support.
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